Molecules that induce disease resistance and improve growth in plants

ABSTRACT

Described herein are methods and compositions for enhancing pathogen immunity in plants and improving plant growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/205,134, filed Mar. 11, 2014, which claims priority to U.S.Provisional Appl. No. 61/777,931 filed Mar. 12, 2013, the disclosures ofeach are incorporated by reference herein in their entireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Nos.DGE0504249 and IOB0449439 awarded by the National Science Foundation.The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Plant innate immunity against pathogens depends on a network offunctionally interconnected genes involved in the regulation andexecution of defense reactions (Glazebrook et al. (2003) Plant J.34:217; Tsuda et al. (2009) PLoS Genet. 5:e1000772). A fundamental formof innate immunity in plants involves conserved microbe-associatedmolecular patterns (MAMPs) or general elicitors. MAMPs are recognized bypattern recognition receptors (PRRs) on the surface of plant cells. Uponpathogen recognition, PRRs activate a comprehensive set of defensereactions collectively referred to as pattern triggered immunity (PTI).Some pathogens have independently acquired the ability to evade PTIthrough the release of effector molecules, suppressing defense and thusenabling infection (effector triggered susceptibility, ETS). In thiscase, the pathogen is virulent and the host susceptible. Even in theface of ETS, plants can mount a weakened immune response, called basaldefense, which limits pathogen spread. Basal defense typically cannotfully prevent disease. As a countermeasure to ETS, plants have alsoevolved the ability to recognize the presence of effectors by highlyspecific plant resistance (R) proteins, which mediate effector triggeredimmunity (ETI) resulting in incompatible interactions and leavingpathogens avirulent.

ETI, basal defense, and PTI pathways share some signaling components,such as reactive oxygen species, Ca²⁺, salicylic acid (SA) and jasmonicacid (JA) (Nimchuk et al. (2003) Ann. Rev. Genet. 37:579). The plantimmune system can be subdivided into various defined sectors that caninteract with each other. For example, distinct defense signalingsectors depend on early MAMP-activated MAP kinases or the messengermolecules SA or JA, and some of these sectors can interact in anadditive or synergistic fashion during PTI, or in an antagonistic mannerduring ETI. The latter phenomenon can compensate if a defined sector isdisabled due to interferences with a pathogen effector. A general reviewof chemical defense inducers can be found in Schreiber & Desveaux (2008)Plant Pathology J. 24:245.

Pesticides are commonly used in agriculture and horticulture for diseasecontrol. Current chemical pesticides, however, typically rely on directantibiotic or biocidal activity, which often leads to undesirable toxicenvironmental side effects. For example, seven of the 10 most frequentlyused pesticides in California tomato production have potential acutetoxic, carcinogenic, neurotoxic or groundwater-contaminating activities,or detrimental effects on human reproduction and development.

BRIEF SUMMARY OF THE INVENTION

Described herein is a class of synthetic elicitors for use inreduced-risk pathogen resistance. Without intending to limit theinvention, it is believed that synthetic elicitors fight plant diseasesby enhancing the plant's inherent defense system. We have surprisinglyfound that the compounds described herein enhance growth of root andaerial structures in plants, pointing to a multifunctional class ofcompounds for improved protection and growth of plants.

Provided herein is a class of multifunctional compounds having a2-phenyl-thiazolidine-carboxylic acid (PTC) structure, including thoseof Formula I:

or a salt thereof, wherein:

-   R¹, R², R³, R⁴, and R⁵ are each independently selected from    hydrogen, halogen, hydroxyl, alkoxyl, and substituted (e.g.,    carboxyl substituted) or unsubstituted heterocycloalkyl (e.g.,    thiazolidine). Such compounds, when contacted with a plant, increase    pathogen resistance or growth of the plant compared to pathogen    resistance or growth of a control plant not contacted with the    compound.

In some embodiments, R¹ is hydrogen or hydroxyl. In some embodiments, R²is hydrogen or bromo. In some embodiments, R³ is hydrogen or R³ is acarboxyl-substituted thiazolidine. In some embodiments, R⁴ is hydrogenor bromo. In some embodiments, R⁵ is hydrogen or methoxy. In someembodiments, R¹ is hydroxyl and R⁴ is hydrogen or bromo. In someembodiments, R² is bromo and R⁴ is hydrogen or methoxy. In someembodiments, the compound is selected from the group consisting of

Further provided is an agricultural composition comprising the compoundas described above formulated for application to a plant or plant part.In some embodiments, the agricultural composition also comprises atleast one of an herbicide, an herbicide safener, a surfactant, afungicide, a pesticide, a nematicide, a plant activator, a synergist, aplant growth regulator, an insect repellant, an acaricide, amolluscicide, or a fertilizer. In some embodiments, the agriculturalcomposition is formulated for spraying or soaking In some embodiments,the agricultural composition is formulated in a dry form, e.g., fordusting or soil application. In some embodiments, the agriculturalcomposition is formulated in a dried or concentrated form to berehydrated or diluted before application.

Further provided are methods of increasing pathogen resistance in aplant, comprising contacting (or applying to) the plant an effectiveamount of a compound as described above, e.g., in an agriculturalcomposition, wherein the compound increases pathogen resistance in theplant compared to pathogen resistance in a plant not treated with thecompound (e.g., an untreated control plant, or the same plant prior tocontacting). In some embodiments, the compound increases pathogenresistance by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% ormore, e.g., as measured by increased pathogen response gene expressionor by reduction in pathogen amount, number, or effect. In someembodiments, the compound reduces the amount or number of pathogen, orreduces the effect of pathogen, at least 1.5-fold, 2-fold, 3-fold,5-fold, 10-fold or more. In some embodiments, the compound is applied ata concentration of 0.5-200 uM, 1-100 uM, 10-100 uM, 10 uM, 25 uM, 50 uM,75 uM, or 100 uM. In some embodiments, the compound is applied more thanonce, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In someembodiments, the compound is applied every 8 hours, twice daily, daily,every other day, twice weekly, etc. In some embodiments, the compound iscontacted with a plant where the pathogen is already present. In someembodiments, the compound is contacted with a plant that is not affectedby pathogen. In some embodiments, the method further comprises detectingthe amount, number or effect of pathogen before contacting, and in someembodiments, further comprises detecting the amount, number or effect ofpathogen after contacting one or more times. In some embodiments, thecompound is contacted with the plant until the pathogen is notdetectable, or is detectable at a level that does not affect the plant.

Further provided are methods of increasing growth of a plant, comprisingcontacting (or applying to) the plant an effective amount of a compoundas described above, e.g., in an agricultural composition, wherein thecompound increases growth of the plant compared to growth of a plant nottreated with the compound (e.g., an untreated control plant grown insimilar conditions). In some embodiments, the compound increases growthby at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more, e.g.,as measured by size or weight of a plant structure. In some embodiments,the compound is applied at a concentration of 0.5-100 uM, 1-100 uM,0.1-10 uM, 0.5-50 uM, 1 uM, 5uM, 10 uM, 25 uM, 50 uM, 75 uM, or 100 uM.In some embodiments, the compound is applied more than once, e.g., atleast 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, thecompound is applied every 8 hours, twice daily, daily, every other day,twice weekly, etc. In some embodiments, the compound is contacted with aplant where pathogen is present.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Analysis of HTC activity under saturation treatment conditions.A.

Screening plate containing 7d-old liquid-grown CaBP22⁻³³³ : :GUSseedlings after x-gluc histochemical staining comparing reporterresponses after 24 h incubation at indicated compound concentrations.The line above wells (1-100 uM) marks GUS stained seedlings. B. Trypanblue staining of CaBP22⁻³³³ : :GUS seedlings incubated for 24 h inmedium containing compounds at the indicated concentrations. Darkness ofthe cotyledons indicates cell death (toxicity) and is pointed to byarrows. The seed coats of seedlings are always darkly stained and canbeen seen in some images. All histochemical staining analyses wereperformed at least three times with similar results.

FIG. 2. Kinetic analysis of chemically-induced disease resistance.Experiments were conducted with three-week-old soil-grown Col-0seedlings sprayed with 100 μM HTC, DCA, INA, or mock solution (1% DMSO)at the indicated times prior to being sprayed with 2×10⁴ mL⁻¹Noco2spores (2 ml per pot). Peronospora (Hyeloperonospora arabidopsidis)spores were counted 7 dpi (days post-infection). Mean and SE values werecalculated from a minimum of three biological replicates and the averageof those is shown above. The Student's t-test (p<0.05) showedsignificant differences for all of the synthetic elicitor treatmentsrelative to the mock-treated control, except for 6 dpt (dayspost-treatment) with HTC.

FIG. 3. Structure-activity analysis of HTC analogs. A. Chemicalstructures of DCA, HTC and tested HTC analogs. Chiral centers of the HTCskeleton are indicated by “1*” and “2*” in HTC. B. Noco2 sporeinhibition assay. Three-week-old soil-grown Col-0 seedlings werespray-infected 24 h after treating with varying concentrations of eachsynthetic elicitor and then assayed at 7 dpi for spore growth. 100%inhibition=0 spores. The assay was repeated three times with similarresults. The average of those three replicates is shown. Significantdifferences of compound-treated compared to mock-treated seedlingsdetermined by Student's t-tests (p<0.05) are marked by colored fourpointed stars.

FIG. 4. Analysis of HTC activity in defense mutant plants. A & B. RT-PCRanalysis of CaBP22 transcript levels in Col-0 (wild-type) and mutantbackgrounds 24 h after spraying 2-week-old seedlings with mock-solution(1% DMSO), 100 μM DCA, or HTC. RT-PCRs with ACTIN are shown as loadingcontrols. At least three biological replicates showed consistentresults. C. Analysis of HTC activity in Col-0 or nahG background.Experiments were conducted with three-week-old soil-grown Col-0 or nahGseedlings sprayed with 100 μM HTC, 100 μM DCA, or mock-solution (1%DMSO) 24 h prior to infection with 2×10⁴ virulent Noco2 spores ml⁻¹ (2ml per pot). Spores were counted at 7 dpi. Mean and SE values werecalculated from a minimum of three biological replicates and theiraverages of are shown. The Student's t-test (p<0.05) showed that HTCsignificantly suppresses Noco2 spore formation in Col-0 but not nahG,while DCA significantly suppresses Noco2 spore formation in bothbackgrounds (marked by stars).

FIG. 5. Relative root length of Col-0 plants grown on syntheticelicitors. Col-0 seedlings were plated in square petri dishes on ½ MSmedium containing 1, 10, 25, 50, 75 or 100 μM of HTC, DCA, INA, or therespective controls (solvent only). A. Roots of Col-0 seedlings grown on1 uM HTC or control (0.01% DMSO) medium 14 days after germination. B.Average percent Col-0 root length grown on synthetic elicitors comparedto Col-0 grown on control. Root length was measured at day 3, 5, 7, 11,and 14. Results are expressed as percentages of the average root lengthon the respective synthetic elicitor treatments compared to theircontrols. The average, standard error, and Student's t-test (p<0.05)were calculated from these values. Significant differences betweensynthetic elicitor-treated and control-treated plants are marked by astar above the corresponding data point. Y-axis represents relative rootgrowth in the presence of synthetic elicitor. C_(SE): concentration ofsynthetic elicitor; dpg: days post germination.

FIG. 6. Relative weight of aerial portions of synthetic elicitor treatedCol-0 seedlings compared to control (DMSO) treated seedlings. Col-0seeds (15-20 seeds per pot) were sown on soil drenched with 20 mL of theindicated concentrations of HTC, INA, DCA, or the respective mocksolutions. Fresh weight (FW) of the above-ground plant tissues wasmeasured at day 14. Results are expressed as percentages of the averageFW of the respective synthetic elicitor treated plants compared to thatof mock-treated controls. The standard error and Student's t-test(p<0.05) were calculated from at least three biological replicates andaverages are shown. Significant differences between syntheticelicitor-treated and control-treated plants are marked by a star abovethe corresponding data point. Y-axis represents percent of FW in thepresence of synthetic elicitor compared to controls.

FIG. 7. Relative root length and FW of tomato seedlings and shoots.Tomato cv. Moneymaker seedlings treated with HTC compared to seedlingstreated with control (DMSO 0.01%). A. Moneymaker seedlings were platedin square petri dishes on ½ MS medium containing HTC. Root length wasmeasured at day 2, 4, 6, and 8. Results are expressed as percentages ofthe average root length on 1 μM HTC or control. The average, standarderror, and Student's t-test (p<0.05) were calculated from these values.Each time point represents at least 89 distinct root length measurementsfrom at least three biological replicates. Y-axis represents percent ofroot growth in the presence of synthetic elicitor compared to controls.B. Moneymaker seeds (five seeds per pot with a minimum of 21 pots) weresown on soil drenched with 20 ml of the indicated concentrations of HTCor their respective control. Fresh weight (FW) of the above-ground planttissues was measured at day 14. Results are expressed as percentages ofthe average weight of HTC treatments compared to those of the controls.The standard error and Student's t-test (p<0.01) were calculated from atleast three biological replicates and the average is shown above.Significant differences between synthetic elicitor-treated andcontrol-treated plants are marked by stars above the corresponding datapoints. Y-axis represents percent of weight in the presence of HTCcompared to controls.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

Synthetic elicitors are drug-like compounds that induce the plantnatural immune response to pathogen. Synthetic elicitors need not betoxic for pathogenic organisms, which allows for production of apathogen defense treatment that is less harmful to humans and theenvironment. Using high-throughput chemical screening, we identifiedsynthetic elicitor candidates that activate expression of thepathogen-responsive CaBP22⁻³³³::GUS reporter gene in transgenicArabidopsis thaliana plants. One of these compounds,2-(5-bromo-2-hydroxy-phenyl)-thiazolidine-4-carboxylic acid (HTC), isable to quickly and transiently induce disease resistance in A. thalianaand is structurally distinct from other known plant defense inducingchemicals. Surprisingly we found that HTC can enhance growth of rootsand aerial parts of A. thaliana and tomato (Solanum lycopersicum). HTCbeneficially affects both plant immunity and growth, thus providing aclass of multi-functional agrochemicals. Moreover, several compoundsthat are structurally related to HTC (2-phenyl-thiazolidine-carboxylicacid (PTC) structures) are shown to have similar activity. Accordingly,provided herein is a class of structurally related compounds that canimprove plant pathogen resistance and improve plant growth.

II. Definitions

An “agricultural composition” is a composition formulated forapplication to a plant or plant part (e.g., seed, cutting, shoots,etc.). An agricultural composition is typically in liquid form, e.g.,for application by spraying or soaking, but can be in a powder forrehydration or application (dusting or dry coating), or gaseous form(e.g., for enclosed environments). The agricultural composition can beconcentrated, e.g., for dilution or water or other solvent. Anagricultural composition can also include more than one activeingredient, e.g., an HTC class compound in combination with a fungicide,herbicide, fertilizer, etc.

The terms “increase pathogen resistance,” “enhance plant immunity,”“promote disease resistance,” “induce plant pathogen defense,” “improveimmunity to pathogens,” and like terms refer to the ability of asubstance to protect a plant from pathogen infection or infestation. Theincrease in protective effect is typically determined by comparison to acontrol. An agent or composition that increases pathogen resistancetypically reduces the number/ amount of pathogen affecting a plant at agiven time post-infection by at least 1.5-fold, e.g., 2-fold, 3-fold,5-fold, 10-fold, or more (or by 20%, 40%, 50%, 70%, 80%, 90% or more)compared to control not treated with the agent or composition. Anincrease in pathogen resistance can be measured in expression ofpathogen-resistance genes (e.g., CaBP22, genes involved in salicylicacid synthesis, etc.) in an affected plant. An agent or composition thatincreases pathogen resistance can lead to an increase of a pathogenresistance gene by at least 1.5-fold, e.g., 2-fold, 3-fold, 5-fold,10-fold, or more (or by 25%, 50%, 75%, 100% or more) compared to controlnot treated with the agent or composition. Pathogen resistance can alsobe determined by observing plant survival time, physical effects ofpathogen invasion (e.g., lesions, stunted or abnormal growth, etc.).Thus, observing or quantifying plant health or the effects of pathogenon a plant can be determinative of the amount or number of pathogenpresent.

The terms “increase growth,” “improve growth,” “enhance growth,”“promote growth,” “induce growth,” and like terms refer to the abilityof a substance to speed up growth of a plant or plant structure over agiven time span, typically as compared to a control. An agent orcomposition that increases growth typically increases the size or weightof a given plant structure, at a given time, by at least 10%, 20%, 40%,50%, 70%, 80%, 90% or more compared to control not treated with theagent or composition.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a compound known to have the desiredeffect (positive control). A control can also represent an average valuegathered from a number of tests or results. One of skill in the art willrecognize that controls can be designed for assessment of any number ofparameters. For example, a control can be devised to compare benefit,e.g., for peripheral composition considerations (e.g., half-life,adhesiveness) or for measures of the desired activity (e.g., comparisonof pathogen resistance, growth, and/or side effects). Controls can bedesigned for in vitro applications, e.g., using reporter gene assays.One of skill in the art will understand which controls are valuable in agiven situation and be able to analyze data based on comparisons tocontrol values. Controls are also valuable for determining thesignificance of data. For example, if values for a given parameter varyin controls, variation in test samples will not be considered assignificant.

Examples of negative controls in the context of the present disclosureinclude plants that are not treated with a particular composition, aplant before treatment, an average value of similar plants grown insimilar but untreated conditions. Examples of positive controls includeplants that are genetically modified for pathogen resistance or rapidgrowth, plants treated with a substance that is known to be toxic to thepathogen in question or known to increase pathogen resistance in aplant. One of skill in the art will understand how to select anappropriate control for a given condition.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. Alkyl can include anynumber of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec.butyl, tert.butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 20 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted.

“Alkylene” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated, and linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkylene can be linked to the same atom ordifferent atoms of the alkylene group. For instance, a straight chainalkylene can be the bivalent radical of —(CH₂)_(n)—, where n is 1, 2, 3,4, 5 or 6. Representative alkylene groups include, but are not limitedto, methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, pentylene and hexylene. Alkylene groups can be substitutedor unsubstituted.

“Alkenyl” refers to a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one double bond. Alkenyl can includeany number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈,C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆.Alkenyl groups can have any suitable number of double bonds, including,but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groupsinclude, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl,1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl,isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl,2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be substitutedor unsubstituted.

“Alkoxy” refers to an alkyl group having an oxygen atom that connectsthe alkyl group to the point of attachment: alkyl-O—. As for alkylgroup, alkoxy groups can have any suitable number of carbon atoms, suchas C₁₋₆. Alkoxy groups include, for example, methoxy, ethoxy, propoxy,iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,pentoxy, hexoxy, etc. The alkoxy groups can be further substituted witha variety of substituents described within. Alkoxy groups can besubstituted or unsubstituted.

“Amine” refers to an —N(R)₂ group where the R groups can be hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, among others. The R groups can be the same or different. Theamino groups can be primary (each R is hydrogen), secondary (one R ishydrogen) or tertiary (each R is other than hydrogen).

“Aryl” refers to an aromatic ring system having any suitable number ofring atoms and any suitable number of rings. Aryl groups can include anysuitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ringmembers. Aryl groups can be monocyclic, fused to form bicyclic ortricyclic groups, or linked by a bond to form a biaryl group.Representative aryl groups include phenyl, naphthyl and biphenyl. Otheraryl groups include benzyl, having a methylene linking group. Some arylgroups have from 6 to 12 ring members, such as phenyl, naphthyl orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl. Some other aryl groups have 6 ring members, such asphenyl. Aryl groups can be substituted or unsubstituted.

“Heterocycloalkyl” refers to a saturated ring system having from 3 to 12ring members and from 1 to 4 heteroatoms of N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can also be oxidized, such as, but not limitedto, —S(O)— and —S(O)₂-. Heterocycloalkyl groups can include any numberof ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8,6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitablenumber of heteroatoms can be included in the heterocycloalkyl groups,such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3to 4. The heterocycloalkyl group can include groups such as aziridine,azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine,pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers),oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane,thiirane, thietane, thiolane (tetrahydrothiophene), thiane(tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine,isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine,dioxane, or dithiane. The heterocycloalkyl groups can also be fused toaromatic or non-aromatic ring systems to form members including, but notlimited to, indoline. Heterocycloalkyl groups can be unsubstituted orsubstituted. For example, heterocycloalkyl groups can be substitutedwith C₁₋₆ alkyl or oxo (═O), among many others.

The heterocycloalkyl groups can be at any position on the ring. Forexample, aziridine can be 1- or 2-aziridine, azetidine can be 1- or 2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine can be1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine,piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1-or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine,isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or 5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

When a heterocycloalkyl includes 3 to 8 ring members and 1 to 3heteroatoms, representative members include, but are not limited to,pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene,thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine,isoxzoalidine, thiazolidine, isothiazolidine, morpholine,thiomorpholine, dioxane and dithiane. Heterocycloalkyl can also form aring having 5 to 6 ring members and 1 to 2 heteroatoms, withrepresentative members including, but not limited to, pyrrolidine,piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine,imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine,isothiazolidine, and morpholine.

“Heterocyclalkylene” refers to a heterocyclalkyl group, as definedabove, linking at least two other groups. The two moieties linked to theheterocyclalkylene can be linked to the same atom or different atoms ofthe heterocyclalkylene. Heterocycloalkylene groups can be substituted orunsubstituted. As used herein, the term unsubstituted indicates theheterocycloalkyl has a full complement of hydrogens, i.e., commensuratewith its saturation level, with no substitutions, e.g., linear decane(—(CH₂)₉—CH₃).

The term halide or halogen refers to the fluorine, chlorine, bromine,and iodine.

The term “hydroxyl” as used herein is represented by the formula —OH.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer, diastereomer, and meso compound,and a mixture of isomers, such as a racemic or scalemic mixture.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, and the like), and progeny of same. Plants that can be treated asdescribed herein include angiosperms (monocotyledonous anddicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes,lycophytes, bryophytes, and multicellular algae.

The term “plant” also includes naturally-occurring mutants,genetically-modified plants, and transgenic plants. A“genetically-modified plant” is one whose genome has been manipulated sothat it is different than a wild-type plant of the same species, varietyor cultivar, e.g., to add a gene or genetic element, remove a gene orgenetic element, change chromatin structure, change RNA expressionlevels, etc. Genetically-modified plants include transgenic plants. A“transgenic plant” refers to a plant that contains genetic material notfound in a wild-type plant of the same species, variety or cultivar. Thegenetic material can include a transgene, a reporter construct, aninsertional mutagenesis event (such as by transposon or T-DNAinsertional mutagenesis), an activation tagging sequence, a mutatedsequence, a homologous recombination event or a sequence modified bychimeraplasty. A transgenic plant may contain an expression vector orcassette. The expression cassette typically comprises apolypeptide-encoding sequence or a modulating nucleic acid (e.g., anantisense, an siRNA or ribozyme) operably linked (i.e., under regulatorycontrol of) to an appropriate inducible or constitutive regulatorysequences that allow for the expression of a polypeptide or modulatingnucleic acid. The expression cassette can be introduced into a plant bytransformation or by breeding after transformation of a parent plant.Such methods can be used in a whole plant, including seedlings andmature plants, as well as to a plant part, such as seed, fruit, leaf, orroot, plant tissue, plant cells or any other plant material, e.g., aplant explant, as well as to progeny thereof, and to in vitro systemsthat mimic biochemical or cellular components or processes in a cell.For example, in one embodiment, the disclosure provides an expressioncassette comprising a pathogen response gene (e.g., CaBP22) promoterregion operably linked to a heterologous polynucleotide, e.g., todetermine if a given compound activates pathogen resistance pathways ina plant. The expression cassette can be used in an expression system,whereby induction of transcription by the promoter can be induced bycontact with a compound of Formula I. Accordingly, transgenic plantscomprising an expression cassette of the disclosure can be induced toexpress a desired gene or polynucleotide upon contact with a compound ofFormula I.

III. Plants and Plant Pathogens

The presently described compounds can be effective for enhancingpathogen immunity and stimulating growth in a broad range of plants,e.g., dicots or monocots, and plants used for food, fiber, or energyproduction. Exemplary plant species include but are not limited tospecies from the genera Asparagus, Atropa, Avena, Brassica, Citrus,Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine,Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca,Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago,Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus,Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella,Triticum, Vitis, Vigna, and, Zea. In some embodiments, the plant is anornamental plant. In some embodiments, the plant is a vegetable- orfruit-producing plant, e.g., tomato, strawberry, fruit tree, etc. Insome embodiments, the presently described compounds are applied to aplant selected from: apple, apricot, avocado, banana, blueberry,boysenberry, Brassicacea crop (e.g., cabbage, cauliflower, rape),carrot, citrus (e.g., orange, tangerine, lemon, grapefruit), cereal crop(e.g., rice, maize, wheat, barley, millet, sorghum, oats, triticale,rye), non-cereal grasses (e.g., bamboo, switch grass), cherry, date,fig, grape, kiwifruit, legume crop (e.g., bean, soybean, pea, cowpea,lentil), marijuana, nectarine, nut, olive, peach, pear, plum, raspberry,solanecea crop (e.g., tobacco, tomato, pepper, potato), strawberry,sugarbeet, sugarcane, and wood crops (e.g., birch, pine, poplar, oak,etc.).

Those of skill will recognize that a number of plant species can be usedas models to predict the effects of the presently described compounds inother plants. For example, it is well recognized that tomato (Solanum)and Arabidopsis plants are useful models, e.g. for other dicots, and Zeacan be a useful model for monocots in particular.

The presently described compounds enhance the immune response of thetreated plant, and thus are believed to be effective against a varietyof plant pathogens, e.g., bacteria, fungi, protists, and viruses.Exemplary pathogens include, but are not limited to, Colletotrichumgraminocola, Diplodia maydis, Verticillium dahliae, Fusariumgraminearum, Fusarium oxysporum and Fusarium verticillioides. Thepresently disclosed compounds can be used to address pathogens thataffect major crops, including: Soybeans: Phytophthora megasperma fsp.glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotiniasclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae(Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotiumrolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica,Colletotrichum dematium (Colletotichum truncatum), Corynesporacassuicola, Septoria glycines, Phyllosticta sojicola, Alternariaalternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestrisp.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophoragregata, Glomerella glycines, Phakopsora pachyrhizi, Pythiumaphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani;Canola: Albugo candida, Alternaria brassicae, Leptosphaeria maculans,Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerellabrassicicola, Pythium ultimum, Hyeloperonospora arabidopsidis(Peronospora parasitica), Fusarium roseum, Alternaria alternata;Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum,Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythiumaphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phomamedicaginis var. medicaginis, Cercospora medicaginis, Pseudopezizamedicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticilliumalbo-atrum, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches,Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii,Leptosphaerulina briosiana, Uromyces striates, Sclerotinia trifoliorum,Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis;Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri,Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v.syringae, Alternaria alternata, Cladosporium herbarum, Fusariumgraminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici,Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola,Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici,Puccinia recondite f.sp. tritici, Puccinia striiformis, Pyrenophoratritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae,Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctoniacerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Clavicepspurpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletiaindica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola,Pythium aphanidermatum; Sunflower: Plasmopora halstedii, Sclerotiniasclerotiorum, Septoria helianthi, Phomopsis helianthi, Alternariahelianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii,Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae,Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticilliumdahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium,Phytophthora cryptogea, Albugo tragopogonis; Corn: Colletotrichumgraminicola, Fusarium verticillioides var. subglutinans, Erwiniastewartii, F. verticillioides, Gibberella zeae (Fusarium graminearum),Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythiumdebaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum,Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T(Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III(Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganense subsp.nebraskense, Trichoderma viride, Claviceps sorghi, Pseudomonas avenae,Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stuntspiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium; Sorghum: Exserohilum turcicum, C. sublineolum, Cercosporasorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringaep.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconiacircinate, Fusarium verticillioides, Alternaria alternate, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisoriumreilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisoriumsorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum,Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerosporaphilippinensis, Sclerospora graminicola, Fusarium graminearum, Fusariumoxysporum, Pythium arrhenomanes, Pythium graminicola, etc.

IV. Compounds

A class of synthetic elicitors useful in the methods described hereinincludes compounds represented by Formula I:

or a salt thereof.

In Formula I, R¹, R², R³, R⁴, and R⁵ are each independently selectedfrom hydrogen, halogen, hydroxyl, alkoxyl, amino, and substituted orunsubstituted heterocycloalkyl. Optionally, R¹ is hydrogen or hydroxyl.Optionally, R² is hydrogen or bromo. Optionally, R³ is hydrogen or asubstituted or unsubstituted heterocycloalkyl (e.g., a substitutedthiazolidine, such as a carboxyl-substituted thiazolidine). Optionally,R⁴ is hydrogen or bromo. Optionally, R⁵ is hydrogen or methoxy.

Examples of Formula I include the following compounds:

The compounds described herein include those with a2-phenyl-thiazolidine-carboxylic acid (PTC) skeleton. In some cases, thecompounds are commercially available (e.g., fromSigma-Aldrich®/TimTec®), or can be prepared using synthetic methodsknown in the art. The compounds described herein can be prepared fromreadily available starting materials. Optimum reaction conditions mayvary with the particular reactants or solvent used, but such conditionscan be determined by one skilled in the art by routine optimizationprocedures.

Variations on Formula I include the addition, subtraction, or movementof the various constituents as described for each compound. Similarly,when one or more chiral centers is present, the chirality of themolecule can be changed. Additionally, compound synthesis can involvethe protection and deprotection of various chemical groups. The use ofprotection and deprotection, and the selection of appropriate protectinggroups can be readily determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Wuts and

Greene, Protective Groups in Organic Synthesis, 4^(th) Ed., Wiley &Sons, 2006. Testing of synthesized compounds of Formula I can be carriedout using reporter assays or pathogen protection assays, e.g., asdescribed in the Examples.

Reactions to produce the compounds described herein can be carried outin one or more solvents, which can be readily selected by one of skillin the art of organic synthesis. Solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products under the conditions at which the reactions are carried out,i.e., temperature and pressure. Product or intermediate formation can bemonitored according to any suitable method, e.g., by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C)infrared spectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Compounds according to Formula I can be prepared, e.g., as described inExample 5, or according to Scheme 1.

As shown in Scheme 1, the compound according to Formula I can be made,for example, by treating an aldehyde (1) with a cysteine hydrochloride(2) to form the compound according to Formula I. Optionally, thecysteine reagent can be a chiral molecule (e.g., L-cysteinehydrochloride) to form a compound of Formula I having chiral centers.Optional chiral centers in Formula I are indicated with asterisks inScheme 1.

In some embodiments, the disclosure provides compositions for protectinga plant from a pathogen comprising an effective amount of at least onecompound of Formula I (e.g., HTC, CMP140, CMP254, CMP023, CMP389,CMP492). In some embodiments, the application of the compositionincreases the expression of a plant pathogen responsive gene (e.g.,CaBP22). “Effective amount” is intended to mean a compound orcomposition sufficient to reduce pathogen survival or growth, e.g., by10%, 20%, 50%, 75%, 80%, 90%, 95%, or more compared to a negativecontrol. In some embodiments, the effective amount of a compound ofFormula I for protecting a plant from a pathogen is 0.05-250 uM, 0.1-200uM, 0.1-100 uM, 0.5-100 uM, 10-100 uM, 0.5-50 uM, or 25-75 uM. Acompound of the disclosure can be applied to a plant or plant part,e.g., the environment of the pathogen, by methods known to those ofordinary skill in the art, including those methods described herein.

In some embodiments, the disclosure provides compounds for increasingplant growth (e.g., roots, leaves, stems, etc.) comprising an effectiveamount of a compound of Formula I (e.g., HTC CMP140, CMP254, CMP023,CMP389, CMP492). “Effective amount” is intended to mean a compound orcomposition sufficient increase growth, e.g., by 10%, 20%, 50%, 75%,80%, 90%, 100%, 150%, 200%, or more, compared to a negative control. Insome embodiments, the effective amount of a compound of Formula I forincreasing plant growth is 0.05-20 uM, 0.1-50 uM, 0.1-10 uM, 0.5-100 uM,1-10 uM, 0.5-50 uM, or 0.1-1 uM. A compound of the disclosure can beapplied to the plant or plant part by methods known to those of ordinaryskill in the art, including those methods described herein.

V. Methods of Screening

Provided herein are methods to determine whether, and to what extent, anHTC-related compound (e.g., a compound of Formula I or a compound with a2-phenyl-thiazolidine-carboxylic acid (PTC) skeleton) can be used toincrease pathogen resistance or increase plant growth.

Assays for determining pathogen resistance are described herein, andinclude reporter assays, e.g., using a reporter construct comprising aregulatory region from a pathogen resistance gene (e.g. a gene on asalicylic acid-dependent pathway, or as described in Knoth et al. (2009)Plant Physiol. 150:333) operatively linked to a reporter (e.g., GFP,luciferase, or other detectable marker). Pathogen resistance can also betested in situ, e.g., by contacting a plant with a pathogen in thepresence or absence (negative control) of a test compound (e.g., anHTC-related compound, a compound of Formula I, or a compound with a2-phenyl-thiazolidine-carboxylic acid (PTC) skeleton), and determiningthe pathogen protecting ability of the test compound by measuring thenumber or amount of the pathogen, or the effect of the pathogen on theplant (e.g., lesions, or reduced plant survival). The pathogen can becontacted with the plant at the same time, before, or after the testcompound. In some embodiments, the plant is contacted with the testcompound before and after contact with the pathogen. In someembodiments, the plant is contacted with the pathogen and/or testcompound is contacted with the plant more than once, e.g., to determinethe duration of efficacy of the compound. The assay can also include apositive control, e.g., a plant contacted with HTC. A reduced number,amount, or effect of pathogen compared to a negative control indicatesthe test compound increases pathogen resistance. One of skill willappreciate that methods of screening an HTC-related compound for itspathogen protecting activity can be adjusted, e.g., efficacy can bemeasured in different ways depending on the effect of the pathogen onthe plant of interest. In addition, a plant tissue can be used insteadof a whole plant. In some embodiments, the plant is agenetically-modified plant, e.g., a plant with a mutation in theSA-dependent pathogen signaling pathway (see, e.g., Lu (2009) PlantSignal Behay. 4:713), or as described in Example 4.

Similarly, assays for determining plant growth are described herein andknown in the art. Growth can be measured from germination, in seedlings,in mature plants, in a plant tissue, or in plant cell culture. In someembodiments, the method comprises contacting a plant cell (e.g., in aplant part or whole plant) with a test compound (e.g., an HTC-relatedcompound, a compound of Formula I or a compound with a2-phenyl-thiazolidine-carboxylic acid (PTC) skeleton), and determiningthe growth rate of the plant cell compared to an untreated control.Growth rate can be determined by measuring the size or weight of a plantor plant part, or by determining the number of cells in a culture, at agiven time after treatment. In some embodiments, the growth assayincludes a positive control such as HTC.

VI. Agricultural Compositions

An agricultural composition comprising a compound of Formula I can alsoinclude one or more of: a surface-active agent, an inert carrier, apreservative, a humectant, a feeding stimulant, an attractant, anencapsulating agent, a binder, an emulsifier, a dye, a UV protective, abuffer, a flow agent, a fertilizer, a nitrogen fixation agent,micronutrient donors, or other preparations that influence plant growth.The agricultural composition can also include one or more agrochemicalsincluding: herbicides, insecticides, fungicides, bactericides,nematicides, molluscicides, acaracides, plant growth regulators, harvestaids, and fertilizers, which can also be combined with carriers,surfactants or adjuvants as appropriate for the agrochemical. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g., naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders, or fertilizers. The active ingredients ofthe present disclosure are normally applied in the form of compositionsand can be applied to the crop area, plant, or seed to be treated. Forexample, the compositions of the present disclosure may be appliedduring growth, seeding or storage.

Surface-active agents that can be used with the presently describedcompounds include anionic compounds such as a carboxylate of, forexample, a metal; carboxylate of a long chain fatty acid; anN-acylsarcosinate; mono- or di-esters of phosphoric acid with fattyalcohol ethoxylates or salts of such esters; fatty alcohol sulfates suchas sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetylsulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenolsulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonatessuch as alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates,e.g., butyl-naphthalene sulfonate; salts of sulfonatednaphthalene-formaldehyde condensates; salts of sulfonatedphenol-formaldehyde condensates; more complex sulfonates such as theamide sulfonates, e.g., the sulfonated condensation product of oleicacid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g., thesodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials that can be used with the presentlydescribed compounds include, but are not limited to, inorganic mineralssuch as kaolin, phyllosilicates, carbonates, sulfates, phosphates, orbotanical materials such as cork, powdered corncobs, peanut hulls, ricehulls, and walnut shells.

Herbicides that can be used with the presently described compoundsinclude compounds that kill or inhibit growth or replication of plants,typically a subset of plants that is distinct from the desired plant orcrop. There are several modes of action: ACCase inhibition, carotenoidbiosynthesis inhibition, cell wall synthesis inhibition, ALS inhibition,ESP synthase inhibition, glutamine synthase inhibition, HPPD inhibition,microtubule assembly inhibition, PPO inhibition, etc. Examples ofcommercially available herbicides include One-Time®, MSMA, Corvus®,Volunteer®, Escalade®, Q4®, Raptor®, Acumen®, Sencor®, Bullet®,TopNotch®, Valor®, PastureGard®, glycophosate (Roundup®), DSMA,Break-Up®, Hyvar®, Barricade®, etc. Herbicides can be mixed with“herbicide safeners” to reduce general toxicity of the herbicide, asdescribed, e.g., in Riechers et al. (2010) Plant Physiol. 153:3.

Pesticides (e.g., nematicides, molluscicides, insecticides,miticide/acaricides) can be used in combination with the presentlydisclosed compounds to kill or reduce the population of undesirablepests affecting the plant. Pesticides can also be used with repellantsor pheromones to disrupt mating behavior. Insectides are directed toinsects, and include, e.g., those of botanical origin (e.g., allicin,nicotine, oxymatrine, jasmolin I and II, quassia, rhodojaponin III, andlimonene), carbamate insecticides (e.g., carbaryl, carbofuran,carbosulfan, oxamyl, nitrilacarb, CPMC, EMPC, fenobucarb), fluorineinsecticides, formamidine insecticides, fumigants (e.g., ethylene oxide,methyl bromide, carbon disulfide), chitin synthesis inhibitors,macrocyclic lactone insecticides, neonicotinoid insecticides,organophosphate insectides, urea and thiourea insectides, etc.Nematicides affect nematodes, and include, e.g., organophosphorusnematicides (e.g., diamidafos, fosthiazate, heterophos, phsphamidon,triazophos), fumigant nematicides (e.g., carbon disulfide, methylbromide, methyl iodide), abamectin, carvacrol, carbamate nematicides(e.g., benomyl, oxamyl), etc. Molluscicides are directed to slugs andsnails, and include, e.g., allicin, bromoacetamide, thiocarb,trifenmorph, fentin, copper sulfate, etc. Many pesticides target morethan one type of pest, so that one or two can be selected to targetinsects, mollusks, nematodes, mitogens, etc.

Fertilizers typically provide macro- and micronutrients in a form thatthey can be utilized by the plant, or a plant-associated organism. Theseinclude, e.g., nitrogen, phosphorus, potassium, sulfur, calcium,potassium, boron, chlorine, copper, iron, manganese, molybdenum, zinc,nickel, and selenium. Fertilizers are often tailored to specific soilconditions or for particular crops or plants. Fertilizers that can beused with the presently described compounds include naturally-occurring,modified, concentrated and/ or chemically synthesized materials, e.g.,manure, bone meal, compost, fish meal, wood chips, etc., or can bechemically synthesized, UAN, anhydrous ammonium nitrate, urea, potash,etc. Suppliers include Scott®, SureCrop®, BCF®, RVR®, Gardenline®, andmany others known in the art.

Fungicides are compounds that can kill fungi or inhibit fungal growth orreplication. Fungicides that can be used with the presently disclosedcompounds include contact, translaminar, and systemic fungicides.Examples include sulfur, neem oil, rosemary oil, jojoba, tea tree oil,Bacillus subtilis, Ulocladium, cinnamaldehyde, etc.

The compositions of the disclosure can be in a suitable form for directapplication or as a concentrate of primary composition that requiresdilution with a suitable quantity of water or other diluent beforeapplication. The concentration of a compound of Formula I will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly, the type of plantand pathogen, and in some cases, on the nature of the use, e.g., forplant growth or pathogen protection.

VII. Methods for Treating a Plant

The presently described compounds can be applied to the environment of aplant or plant pathogen by, for example, spraying, atomizing, dusting,scattering, coating or pouring, introducing into or on the soil,introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pathogen has begun to appearor before the appearance of pathogens as a protective measure. Typicallypathogen control is contemplated early in plant growth, as this is thetime when the plant can be most severely damaged. The compositions ofthe disclosure can conveniently contain an insecticide if this isthought necessary.

The presently described compounds can be applied simultaneously or insuccession with other compounds. Methods of applying an activeingredient or agricultural composition of the present disclosure thatcontains at least one compound of Formula I include, but are not limitedto, foliar application (e.g., spray or soak), seed coating, and soilapplication. The number of applications and the rate of applicationdepend on desired use, e.g., pathogen protection or increased growth,and conditions, e.g., the intensity of pathogen infestation or growingconditions.

The examples and embodiments described herein are for illustrativepurposes only, and various modifications or changes are to be includedwithin the spirit and purview of this application and scope of theappended claims. All patents, patent applications, internet sources, andother published reference materials cited in this specification areincorporated herein by reference in their entireties.

VIII. Examples

A. Example 1 HTC is a Small Molecule Elicitor of CaBP22⁻³³³::GUSExpression

We took a chemical genomics-based approach to identify new syntheticelicitors for the plant immune system and develop environmentally safepesticides. By high-throughput chemical screening of commerciallyavailable chemical libraries, we identified drug-like organic compoundsthat induce the pathogen-responsive pCaBP22⁻³³³::GUS reporter gene intransgenic Arabidopsis. We reported one of them,3-5-dicholoroanthranlilic acid (DCA) in Knoth et al. (2009) PlantPhysiol. 150:333. DCA triggered fast, strong and transient diseaseresistance against as the pathogenic oomycete Hyaloperonosporaarabidopsidis (Peronospora) and the bacterial pathogen Pseudomonassyringae. DCA activity was shown in various Arabidopsis defense mutantsto be partially dependent on the WRKY70 transcription factor, incontrast to INA and BTH, which are fully dependent on thetranscriptional co-factor.

HTC was also selected from the compounds identified in the reporterassay screen. HTC was selected from the Sigma-Aldrich®/TimTec®MyriaScreen® library, but has not been reported as a synthetic elicitor,and has a chemical structure distinct from DCA or any other known plantdefense inducer. To quantify the bioactivity of HTC, we performedvarious assays with one week-old CaBP22⁻³³³::GUS seedlings submerged inliquid growth medium containing HTC or mock-solution (saturationtreatment). HTC activated CaBP22⁻³³³::GUS expression within 24 h at aconcentration as low as 1 μM (FIG. 1A). To examine if HTC inducesphytotoxicity, we stained Col-0 seedlings after saturation treatmentwith trypan blue. We observed dark staining indicating cell death in100% of the seedlings treated for 24 h with 300 μM HTC (FIG. 1B). Nocell death was observed at lower concentrations that resulted inreporter activation (1-100 μM), indicating that HTC-inducedphytotoxicity is not responsible for its effect on CaBP22⁻³³³::GUSexpression.

B. Example 2 HTC Causes Rapid and Transient Resistance to Peronospora

We next tested whether HTC could induce pathogen resistance insoil-grown plants. Col-0 sprayed with HTC concentrations as low as 10 μMprior to infection with the virulent Peronospora isolate Noco2 exhibitedsignificantly reduced numbers of Noco2 spores 7 days post infection(dpi). To determine if HTC differs from other synthetic elicitors in thekinetics of defense induction, Col-0 seedlings were sprayed with 100 μMof INA, DCA, HTC, or mock, 1 h, 3 h, 1 d, 3 d, or 6 d prior to pathogenchallenge. The mock solvent treatment diminished spore growth whenpathogen treatment and pre-treatment were less than one day apart,possibly due to liquid coating Arabidopsis seedlings and interferingwith the Peronospora spore suspension.

DCA and INA induced full resistance to Noco2 lh post treatment (hpt).All three synthetic elicitors strongly suppressed Peronospora sporeproduction to similar levels by 3 hpt. FIG. 2 shows that HTC-triggeredimmunity to Noco2 was reduced at 3 days post treatment (dpt), and notdetectable at 6 dpt it was no longer detectable. The results show thatHTC, like DCA, is a fast, potent, but reversible inducer of immunityagainst Noco2.

C. Example 3 Structure-Activity Analysis of HTC

To determine which substituents or moieties of the HTC molecule arecritical for its defense-inducing activity, seven commercially availableHTC analogs were analyzed (FIG. 3A). We compared the ability of thesecompounds to induce GUS expression in saturation treated CaBP22⁻³³³::GUSArabidopsis seedlings to that of DCA and HTC. Except for4-carboxy-4-thiazolidinyl (T4CA) and 5-bromo-2-hydroxy-phenyl (2BP),which represent the two halves of HTC when linked by a covalent bond,all tested HTC analogs induced CaBP22⁻³³³::GUS expression atconcentrations between 1-100 uM. The most efficient HTC analog was2-(2-hydroxy-phenyl)-thiazolidine-4-carboxilic acid (CMP140), whichlacks the bromine substituent of the phenyl ring. We also used trypanblue staining to examine HTC-analog-induced phytotoxicity. Cell death,indicated by staining, was observed in 100% of the seedlings treated for24 h at 300 μM of HTC and DCA. No cell death was observed at anyconcentrations examined for the HTC analogs, indicating that cell deathwas not responsible for CaBP22⁻³³³::GUS activation by these compounds.

We performed a dose-response analysis of inhibition of Noco2 sporedevelopment in three-week old Col-0 plants using the HTC analogs (FIG.3B). The data from the Noco2 defense assays and the reporter gene assaysshowed similar trends. DCA and HTC provided highest protection againstNoco2 infection, significantly protecting Col-0 from s at 10 uM. T4CAand 2BP did not result in significant immunity, except for 2BP at 100uM. CMP 140, which could induce CaBP22⁻³³³::GUS expression, was a lessefficient defense inducer compared to HTC, and a concentration of atleast 100 uM was required to significantly reduce pathogen growth. Allremaining compounds, which are all derivatives of the2-phenyl-thiazolidine-carboxylic acid (PTC) skeleton exhibitedintermediate levels of defense-inducing activity. The results show thatHTC is the most potent of the tested analogs, but the fact that otherPTC derivatives successfully activate immune responses establishes PTCsas a new class of synthetic elicitors.

D. Example 4 HTC is Functionally Distinct from DCA

To establish the mode-of-action of HTC, we performed reversetranscription (RT)-PCRs examining transcript levels of the defensemarker gene CaBP22 in Col-0 and a set of mutant lines representingvarious defense signaling mechanisms. Both DCA and HTC induced anincrease of CaBP22 transcript levels in Col-0 plants, but not in thewrky70-3 mutant (FIG. 4A). In the transgenic nahG line, which iscompromised in the accumulation of salicylic acid (SA), the ability ofHTC to induce CaBP22 expression was blocked, while that of DCA was notaffected (FIGS. 4A and B).

These results show that the mode-of-action of HTC is distinct from thatof DCA and that HTC likely interferes with defense signaling upstream ofSA. Consistently, HTC-induced CaBP22 transcript accumulation is reducedin the pad4-1 and sid2-2 mutants, which are deficient in a distinctpathway of defense-associated SA synthesis (FIGS. 4A and B). Thewrky72-2 mutant, which is likely be deficient in an SA-independent basaldefense mechanism (Bhattarai et al. (2010) Plant J. 63:229), exhibitsfull HTC-induced CaBP22 transcript accumulation (FIG. 4B). Furthermore,DCA induced resistance to Noco2 in both Col-0 and nahG, while HTC wasonly able to do so in Col-0 (FIG. 4C). We did not observe significantreduction of Noco2 spores in nahG mutants after HTC treatment. Theseresults confirm that the mode-of-action of HTC is distinct from that ofDCA.

E. Example 5 Synthesis of HTC

Our studies were performed with HTC purchased from Sigma TimTec®(p-HTC). HTC can, however, be easily synthesized through the reaction ofL-Cysteine hydrochloride with 5-bromo-salicyaldehyde. We synthesized HTC(s-HTC) using the protocols described in Kulaeva et al. (1992) PlantMol. Biol. 20:383 and Song et al. (2009) PNAS 106:1654. Briefly, 2.0 gL-cysteine hydrochloride and 1.0 g NaOAc were dissolved in 17 mL sterileMilliQ® water. 1.0 g 5′bromo-salicyaldehyde dissolved in 18 mL was addedto this solution with constant stirring. The mixture was vigorouslystirred at room temperature overnight. The product was separated bysuction filtration and washed several times with water and then ethanol.

The s-HTC preparation produced a nuclear magnetic resonance (NMR)spectrum identical to that obtained with p-HTC. We also compared thebiological activity of s-HTC to that of p-HTC by spraying aconcentration gradient of both preparations on soil-grown Col-0seedlings 24 h prior to challenge with Noco2. No significant differencewas found between the efficacy of s-HTC and p-HTC in reducing Noco2spore counts at 20, 50, 100, and 200 μM. HTC has two chiral centers(indicated with 1* and 2* in FIG. 3A), but only two conformations of thediastereomers were detected by NMR. NMR analysis further revealed thatthese two diastereomers are present in s-HTC and p-HTC at the same ratio(40% to 60%).

F. Example 6 HTC Induces Growth of Plant Structures

Surprisingly, at low concentrations (1 μM) HTC enhanced the root lengthof Arabidopsis plants grown on ½ MS agar plates (FIG. 5A and B). Asshown in FIG. 1A, this dose is sufficient to induce CaBP22⁻³³³::GUSexpression. Higher doses of HTC resulted in reduced root growth. Wecompared the ability of other synthetic elicitors to enhance growth, andperformed root growth assays with INA, DCA, and HTC (FIG. 5B). Eachsynthetic elicitor was added to the solid media plates at concentrationsranging from 1 to 100 μM. The plates were placed upright in a growthchamber, and root lengths were measured at 3 d-intervals. To merge datasets, root length was expressed as a percentage of their value relativeto the average of the mock (control) for that data set. Normalizing eachroot length to its control allowed all replicates to be combinedtogether to calculate averages, standard errors, and perform statisticaltests (FIG. 5B).

The effect was most dramatic with HTC, which at 1 μM increased rootlength to up to 180% of that observed on control plates, but DCA and INAalso enhanced Arabidopsis root growth. HTC exhibited this rootlengthening activity only at the lowest tested concentration (1 μM), incontrast to DCA and INA, which enhanced root length at various differenttested concentrations. For all three tested synthetic elicitors,enhancement of root growth was detectable within three days ofgermination, but was more pronounced at later time-points. Both HTC andDCA, but not INA, strongly inhibited Arabidopsis root growth at higherconcentrations. Again, this effect was most pronounced with HTC, whichat concentrations above 50 μM reduced the length of Arabidopsis roots by˜80%. The observation that low doses of HTC and DCA stimulate rootgrowth, while high doses inhibit root growth, is reminiscent ofhormesis, a phenomenon characterized by low dose stimulation and highdose inhibition of biological responses.

To determine whether the synthetic inhibitors could increase the aerialweight of Arabidopsis, soil-sown-seedlings were drenched with syntheticelicitor in water at day 0 (FIG. 6). After 14 days, aerial portions ofplants were cut off and their fresh weight measured. Shoots of plantstreated with 1-100 μM HTC showed a significant increase in their freshweight, reaching ˜120% of the values observed with mock-treated plants.HTC thus has a hormesis-like effect in Arabidopsis roots, but generallyincreases the aerial fresh weight of Arabidopsis at the concentrationstested.

We also performed root and shoot growth assays with tomato plants. Weapplied 1 μM HTC to a large set of plate-grown tomato plants (n>89)resulted in a significant increase in root length at all analyzedtime-points (FIG. 7A). The greatest increase was seen at day 6 withHTC-treated tomato reaching 136.1% of the root length of mock-treatedplants. We also performed root drench-assays to determine effects of HTCon aerial plant mass in soil-grown tomato. Tomato seedlings were plantedin soil and drenched a single time with 1, 10, or 25 μM of HTC (FIG.7B). Application of 10 μM HTC caused a significant increase in the massof shoots, resulting in 128.5% of the fresh weight measured inmock-treated plants. The data show that HTC causes an increase in boththe root length and aerial weight of tomato plants.

What is claimed is:
 1. An agricultural composition to be applied to aplant comprising the compound of Formula I

or a salt thereof, wherein: R¹ and R⁵ are each independently selectedfrom hydrogen, hydroxyl, and alkoxyl, R² and R⁴ are each independentlyselected from hydrogen, halogen, R³ is selected from hydrogen andsubstituted or unsubstituted heterocycloalkyl, wherein, when contactedwith a plant, the compound increases pathogen resistance or growth ofthe plant compared to pathogen resistance or growth of a control plantnot contacted with the compound.
 2. The agricultural composition ofclaim 1, wherein R¹ is hydrogen or hydroxyl.
 3. The agriculturalcomposition of claim 1, wherein R² is hydrogen or bromo.
 4. Theagricultural composition of claim 1, wherein R³ is hydrogen.
 5. Theagricultural composition of claim 1, wherein R³ is acarboxyl-substituted thiazolidine.
 6. The agricultural composition ofclaim 1, wherein R⁴ is hydrogen or bromo.
 7. The agriculturalcomposition of claim 1, wherein R⁵ is hydrogen or methoxy.
 8. Theagricultural composition of claim 1, wherein the compound is selectedfrom the group consisting of:


9. The agricultural composition of claim 1, further comprising at leastone of an herbicide, an herbicide safener, a surfactant, a fungicide, apesticide, a nematicide, a plant activator, a synergist, a plant growthregulator, an insect repellant, an acaricide, a molluscicide, or afertilizer.
 10. A method for increasing pathogen resistance in a plant,comprising contacting the plant with an effective amount of a compoundof Formula I,

or a salt thereof, wherein: R¹, R², R³, R⁴, and R⁵ are eachindependently selected from hydrogen, halogen, hydroxyl, alkoxyl, andsubstituted or unsubstituted heterocycloalkyl, thereby increasingpathogen resistance in the plant compared to pathogen resistance of acontrol plant not contacted with the compound.
 11. The method of claim10, wherein the compound is selected from the group consisting of:


12. The method of claim 10, wherein the compound is applied in solutionat a concentration of 10-100 uM.
 13. The method of claim 10, wherein thepathogen is present on or in the plant at the time of the contactingstep.
 14. The method of claim 10, further comprising comparing theamount of pathogen present on the plant before and after the contactingstep.
 15. The method of claim 10, wherein the compound is applied incombination with at least one of an herbicide, an herbicide safener, asurfactant, a fungicide, a pesticide, a nematicide, a plant activator, asynergist, a plant growth regulator, an insect repellant, an acaricide,a molluscicide, or a fertilizer.
 16. A method for increasing growth of aplant, comprising contacting the plant with an effective amount of acompound of Formula I,

or a salt thereof, wherein: R¹, R², R³,R⁴, and R⁵ are each independentlyselected from hydrogen, halogen, hydroxyl, alkoxyl, and substituted orunsubstituted heterocycloalkyl thereby increasing growth of the plantcompared to growth of a control plant not contacted with the compound.17. The method of claim 16, wherein the compound is selected from thegroup consisting of:


18. The method of claim 16, or 17, wherein the compound is applied insolution at a concentration of 0.5-50 uM.
 19. The method of claim 16,wherein a pathogen is present on or in the plant at the time of thecontacting step.
 20. The method of claim 16, wherein the compound isapplied in combination with at least one of an herbicide, an herbicidesafener, a surfactant, a fungicide, a pesticide, a nematicide, a plantactivator, a synergist, a plant growth regulator, an insect repellant,an acaricide, a molluscicide, or a fertilizer.